PTEN hinders the formation of scars by regulating the levels of proteins in the extracellular matrix and promoting the apoptosis of dermal fibroblasts through Bcl-xL.

Hypertrophic scar (HS) is a dermatological condition characterized by an excessive accumulation of proteins in the extracellular matrix (ECM) and an elevated cell count. The development of HS is thought to be linked to the disruption of dermal fibroblast proliferation and apoptosis. The processes of cell proliferation and apoptosis are notably influenced by PTEN. However, the precise mechanisms by which PTEN regulates hypertrophic scar fibroblasts (HSFs) and its overall role in scar formation are still not fully understood. The objective of this study was to investigate the influence of PTEN on hypertrophic scars(HS) and its function in the regulation of scar formation, with the aim of identifying a pivotal molecular target for scar treatment. Our results demonstrate that the overexpression of PTEN (AdPTEN) significantly suppressed the expression of type I collagen (Col I), type III collagen (Col III), and alpha smooth muscle actin (α-SMA) in HSFs. Furthermore, it was observed that the introduction of AdPTEN resulted in the suppression of Bcl-xL expression, which consequently led to an increase in the apoptosis of HSFs. Similarly, in the inhibition of collagens expression and subsequent increase in HSF apoptosis were also observed upon silencing Bcl-xL (sibcl-xL). Additionally, the in vitro model demonstrated that both AdPTEN and sibcl-xL were effective in reducing the contraction of FPCL. The findings of our study provide validation for the role of PTEN in inhibiting the development of hypertrophic scars (HS) by modulating the expression of extracellular matrix (ECM) proteins and promoting apoptosis in hypertrophic scar fibroblasts (HSFs) via Bcl-xL. These results indicate that PTEN and Bcl-xL may hold promise as potential molecular targets for therapeutic interventions aimed at managing hypertrophic scars.

Coacervation of Spherical Polyelectrolyte Brushes with Additional Polyelectrolytes Bearing Positive or Negative Charges.

By combining small-angle X-ray scattering, wide-angle X-ray scattering, and rheology, the effect of additional polyelectrolyte chains on interactions among spherical polyelectrolyte brushes (SPB) was systematically investigated both on microscopic and macroscopic levels. The negatively charged poly(acrylic acid) (PAA) chains and positively charged poly(dimethyl diallyl ammonium chloride) (PDDA) chains were used as additional polyelectrolyte chains to investigate the local ordered structure and the "polyelectrolyte peak" among SPB. Interestingly, coacervation appeared in the SPB emulsion while introducing additional free polyelectrolyte chains. The addition of excess positively charged PDDA chains would lead to the transformation of the SPB emulsion from the coacervation to the aggregation, while it has not been observed in the case of PAA chains. Moreover, it was further confirmed that the specific local ordered structure was caused by the electrostatic interaction among polyelectrolyte chains of adjacent SPB. This work could enrich our understanding of polyelectrolyte assembly in concentrated SPB, thereby greatly broadening the application fields of SPB.

Interaction among Spherical Polyelectrolyte Brushes in Concentrated Aqueous Solution.

Interaction among concentrated spherical polyelectrolyte brushes (SPB) dispersions in water was systematically investigated by means of small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS), and rheological methods. SPB consist of a core of polystyrene (PS) and a poly(acrylic acid) (PAA) brush shell. The "polyelectrolyte peak" appeared in SAXS spectra and was observed in WAXS curves for the first time. The size of the polyelectrolyte peak and the rheological properties of SPB were found to be strongly effected by SPB concentration, pH, and ionic strength. Combined with SAXS, WAXS, and rheological results, it is confirmed that the polyelectrolyte peak is originated from local ordered structures of polyelectrolyte chains bridged by counterions in the overlapping area among SPB driven by electrostatic interactions.

Outcome of elective switching to vedolizumab in inflammatory bowel disease patients under tumor necrosis factor antagonist-maintained clinical remission.

BACKGROUND AND AIM: Vedolizumab (VDZ) has been used in inflammatory bowel disease (IBD) patients who failed anti-tumor necrosis factor (TNF) therapy. This study was to examine long-term outcome of IBD patients switching to VDZ from anti-TNF agents for reasons other than failure of therapy. METHODS: Inflammatory bowel disease patients at the University of Chicago IBD center who were in clinical remission with anti-TNF therapy and then electively changed to VDZ due to reasons other than loss of response were retrospectively analyzed. The primary outcome was the durability of clinical remission maintained by VDZ as assessed by Kaplan-Meier survival analysis. The proportion of patients in clinical and endoscopic remission at 6-12 months after switching to VDZ therapy was analyzed. RESULTS: A total of 41 patients (36 with Crohn's disease and 5 with ulcerative colitis) met the inclusion criteria and were in clinical remission at the time of switch. The majority of patients switched therapy due to adverse effects (56.1%) or infections (14.6%). During a median duration of 30 months (range 7-52) of VDZ therapy, 34 (82.9%) were in VDZ-maintained clinical remission. One (2.4%) and four (9.8%) patients discontinued VDZ due to flare and adverse effects, respectively. Endoscopic remission was present in 25 of 30 patients (83.3%) who had a follow-up colonoscopy. CONCLUSIONS: Vedolizumab was effective and safe in maintaining remission in IBD patients who switched from anti-TNF agents due to reasons other than failure of therapy. Our results suggest that switching anti-TNF remitters to VDZ treatment is a safe practice in specific patient populations.

Distinct roles of intracellular heat shock protein 70 in maintaining gastrointestinal homeostasis.

The inducible heat shock protein 70 (Hsp70) is both cytoprotective and immunomodulatory, potentially accounting for its critical role in maintaining gastrointestinal homeostasis. When levels are reduced in conditions like inflammatory bowel diseases (IBD), loss of function contributes to the severity and chronicity of these diseases, although through which cell types and mechanisms remains unclear. Here, the role of Hsp70-mediated intestinal epithelial protection and immune regulation in experimental colitis was examined by using a villin promoter-driven Hsp70 transgene in the 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models and in IL-10/Hsp70 double knockout (IL10-/-/Hsp70-/-) mice. In addition, Hsp70-mediated IL-10 production and immune protection were investigated using a CD45RBhigh transfer model and measuring colonic and immune cell cytokine expression during colitis. We found that the epithelial-specific expression of Hsp70 transgene attenuated DSS-induced colitis in Hsp70-/- mice by protecting tight junctions (TJ) and their interaction with the TJ-associated protein ZO-1. In the TNBS colitis and CD45RBhigh model, Hsp70 carried out its intracellular anti-inflammatory function by maintaining IL-10 production. Impaired ERK phosphorylation, but not p38 or JNK phosphorylation pathways, was associated with decreased IL-10 production in Hsp70-deficient cells. Together, these actions can be leveraged in the context of cellular specificity to develop complementary strategies that can lead to reduction in mucosal injury and immune activation in colonic colitis development. NEW & NOTEWORTHY Using four different experimental colitis models, we filled an important gap in knowledge by defining essential roles of intracellular heat shock protein 70 in different cell types in maintaining intestinal integrity and immune regulation. These findings are relevant to human inflammatory bowel diseases and represent potential avenues for developing therapeutic strategies, not only to counter the destructive processes of inflammation but also to promote tissue healing and prevent complications frequently associated with chronic intestinal inflammation.

Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/- mice.

The composite human microbiome of Western populations has probably changed over the past century, brought on by new environmental triggers that often have a negative impact on human health. Here we show that consumption of a diet high in saturated (milk-derived) fat, but not polyunsaturated (safflower oil) fat, changes the conditions for microbial assemblage and promotes the expansion of a low-abundance, sulphite-reducing pathobiont, Bilophila wadsworthia. This was associated with a pro-inflammatory T helper type 1 (T(H)1) immune response and increased incidence of colitis in genetically susceptible Il10(−/−), but not wild-type mice. These effects are mediated by milk-derived-fat-promoted taurine conjugation of hepatic bile acids, which increases the availability of organic sulphur used by sulphite-reducing microorganisms like B. wadsworthia. When mice were fed a low-fat diet supplemented with taurocholic acid, but not with glycocholic acid, for example, a bloom of B. wadsworthia and development of colitis were observed in Il10(−/−) mice. Together these data show that dietary fats, by promoting changes in host bile acid composition, can markedly alter conditions for gut microbial assemblage, resulting in dysbiosis that can perturb immune homeostasis. The data provide a plausible mechanistic basis by which Western-type diets high in certain saturated fats might increase the prevalence of complex immune-mediated diseases like inflammatory bowel disease in genetically susceptible hosts.

Core-Shell-Corona Silica Hybrid Nanoparticles Templated by Spherical Polyelectrolyte Brushes: A Study by Small Angle X-ray Scattering.

Core-shell-corona silica/polymer hybrid nanoparticles with narrow size distribution were prepared in the template of spherical polyelectrolyte brushes (SPB) which consist of a solid polystyrene (PS) core densely grafted with linear poly(acrylic acid) (PAA) chains. The microstructure of obtained hybrid nanoparticles was studied by small-angle X-ray scattering (SAXS) and in combination with dynamic light scattering (DLS) and transmission electron microscopy (TEM). The generation of silica shell within the brush is confirmed by the significant increase of the electron density in the shell, and the silica shell showed a unique inner-loose-outer-dense structure, whose thickness is pH sensitive but is insensitive to ionic strength as revealed by fitting SAXS data. After dissolving the PS core, hollow silica nanoparticles were obtained and determined by SAXS, which should be ideal carriers for pH-triggered drug delivery. SAXS is confirmed to be a powerful method to characterize the core-shell-corona silica/polymer hybrid and hollow silica nanoparticles.

Protein Immobilization onto Cationic Spherical Polyelectrolyte Brushes Studied by Small Angle X-ray Scattering.

The immobilization of bovine serum albumins (BSA) onto cationic spherical polyelectrolyte brushes (SPB) consisting of a solid polystyrene (PS) core and a densely grafted poly(2-aminoethyl methacrylate hydrochloride) (PAEMH) shell was studied by small-angle X-ray scattering (SAXS). The observed dynamics of adsorption of BSA onto SPB by time-resolved SAXS can be divided into two stages. In the first stage (tens of milliseconds), the added proteins as in-between bridge instantaneously caused the aggregation of SPB. Then BSA penetrated into the brush layer driven by electrostatic attractions, and reached equilibrium in the second stage (tens of seconds). The amount of BSA immobilized onto brush layer reached the maximum when pH was increased to about 6.1 and BSA concentration to 10 g/L. The cationic SPB were confirmed to provide stronger adsorption capacity for BSA compared to anionic ones.

The Effect of Additives on the Early Stages of Growth of Calcite Single Crystals.

As crystallization processes are often rapid, it can be difficult to monitor their growth mechanisms. In this study, we made use of the fact that crystallization proceeds more slowly in small volumes than in bulk solution to investigate the effects of the soluble additives Mg2+ and poly(styrene sulfonate) (PSS) on the early stages of growth of calcite crystals. Using a "Crystal Hotel" microfluidic device to provide well-defined, nanoliter volumes, we observed that calcite crystals form via an amorphous precursor phase. Surprisingly, the first calcite crystals formed are perfect rhombohedra, and the soluble additives have no influence on the morphology until the crystals reach sizes of 0.1-0.5 µm for Mg2+ and 1-2 µm for PSS. The crystals then continue to grow to develop morphologies characteristic of these additives. These results can be rationalized by considering additive binding to kink sites, which is consistent with crystal growth by a classical mechanism.

Occlusion of Sulfate-Based Diblock Copolymer Nanoparticles within Calcite: Effect of Varying the Surface Density of Anionic Stabilizer Chains.

Polymerization-induced self-assembly (PISA) offers a highly versatile and efficient route to a wide range of organic nanoparticles. In this article, we demonstrate for the first time that poly(ammonium 2-sulfatoethyl methacrylate)-poly(benzyl methacrylate) [PSEM-PBzMA] diblock copolymer nanoparticles can be prepared with either a high or low PSEM stabilizer surface density using either RAFT dispersion polymerization in a 2:1 v/v ethanol/water mixture or RAFT aqueous emulsion polymerization, respectively. We then use these model nanoparticles to gain new insight into a key topic in materials chemistry: the occlusion of organic additives into inorganic crystals. Substantial differences are observed for the extent of occlusion of these two types of anionic nanoparticles into calcite (CaCO3), which serves as a suitable model host crystal. A low PSEM stabilizer surface density leads to uniform nanoparticle occlusion within calcite at up to 7.5% w/w (16% v/v), while minimal occlusion occurs when using nanoparticles with a high PSEM stabilizer surface density. This counter-intuitive observation suggests that an optimum anionic surface density is required for efficient occlusion, which provides a hitherto unexpected design rule for the incorporation of nanoparticles within crystals.

[Study on change rule of 6 ester-type alkaloids in process of Heishunpian].

To study the variation of six ester-type alkaloids and characteristic fingerprints in the process from Radix Aconite Lateralis to Heishunpian and lay a foundation for the study of the processing principle of Heishunpian, HPLC. analysis was performed on a Phenomenex Gemini C18 (4.6 mm x 250 mm, 5 microm) with acetonitrile and 40 mmol x L(-1) ammonium acetate (adjusted to pH 10 with concentrated ammonia water) as mobile phase. The detection wavelength was set at 235 nm. The flow rate was set at 0.8 mL x min(-1) and the injection volume was 10-20 microL. Six ester-type alkaloids were determined and characteristic fingerprints of the process were established. As the process continues, the contents of diester diterpene alkaloids were decreased step by step, while the contents varia tion of monoester diterpene alkaloids were not obvious. Each sample showed significant difference in characteristic fingerprints. With the exception of 6 known monoester diterpene alkaloids and diester diterpene alkaloids, 13 peaks were marked in the characteristic fingerprints, of which the total change rule of the other 7 unknown peaks were similar with 3 diester diterpene alkaloids. The established method is accurate, reliable and repeatable, and can provide reference for revealing change rule of index components and illuminating processing principle in the process of Heishunpian.

Panax notoginseng attenuates experimental colitis in the azoxymethane/dextran sulfate sodium mouse model.

Patients suffering from inflammatory bowel disease are at a high risk of developing colorectal cancer. To assess the anticancer potential of botanicals, in this study, we evaluated the effects of Panax notoginseng on azoxymethane/dextran sulfate sodium (DSS)-induced colitis. One week after A/J mice received azoxymethane, the animals received DSS for 8 days or were supplemented with P. notoginseng extract, at 30 or 90 mg/kg. DSS-induced colitis was scored with the disease activity index. The severity of the inflammatory lesions was evaluated by a colon tissue histological assessment. The expression of inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) were also explored. We observed that the effects of P. notoginseng on the reduction of colon inflammation, expressed in disease activity index score, were in a dose-related manner (p < 0.01). P. notoginseng inhibited the reduction of the colon length and the loss of bodyweight in dose-related manner (all p < 0.05). The histological assessment of the colitis and inflammatory-related immunohistochemical data also supported the pharmacological observations. Our data suggest that P. notoginseng is a promising candidate in preventing and treating colitis and inflammation-associated colon carcinogenesis.

Enhanced Performance and High Resistance to Efficiency Degradation of Blue Quantum-Dot Light-Emitting Diodes Using the Lewis Base Blended Hole-Transporting Layers.

The distinctive characteristics of blue quantum dots (QDs) such as their deep valence band and large bandgap give rise to an elevated hole injection barrier between the hole transport layers (HTLs) and the QD active layer. This results in an imbalance of carrier transport and injection across the device, leading to a degrading performance in QD light-emitting diodes (QLEDs). In this paper, high-efficiency and low-efficiency degradation blue CdSe/CdS/ZnS QLEDs were fabricated by using the Lewis base, 1,2-bis(diphenylphosphino)ethane (DPPE), blended with poly(9-vinylcarbazole) (PVK) (DPPE:PVK) as HTLs. The device performance of blue QLEDs can be finely adjusted by manipulating the blending ratio between DPPE and PVK. When 4 wt % DPPE was blended with PVK (4 wt % DPPE:PVK) as the HTL, the device achieved its optimal performance. Compared to the device with neat PVK as the HTL, the turn-on voltage of blue QLEDs with the 4 wt % DPPE:PVK HTL is reduced from 3.21 to 2.9 V. The maximum current efficiency (CE) and external quantum efficiency (EQE) of blue QLEDs increase from 2.92 cd A-1 and 5.89% in neat PVK to 5.75 cd A-1 and 11.75% for the 4 wt % DPPE:PVK HTL. Furthermore, the QLEDs incorporating DPPE:PVK HTLs exhibited exceptional resistance to efficiency degradation (EQE = 8.83%@L = 12,000 cd m-2 for 4 wt % DPPE:PVK as the HTL and EQE = 2.80%@L = 12,000 cd m-2 for neat PVK as the HTL). A more in-depth analysis reveals that enhanced device performance results from the chelating and bridging effect of the bidentate ligand Lewis base DPPE. These effects strengthen the binding of free metal ions in the blue QDs, reduce the charge barriers, enhance the contact between the HTLs and the QD active layer, and ultimately improve hole injection.

Preparation of an Inorganic All-Solid-State Electrochromic Device with Excellent Open-Circuit Memory.

Due to the spontaneous transport of small-sized cations and redox reactions under open circuit conditions, the currently reported coloring electrochromic devices (ECDs) may self-bleach easily. The resulting ECDs exhibit poor open-circuit memory, which limits their applications in static display advertisement. By constructing energy barriers to effectively control small-sized cation transport, the redox reaction could be suppressed, thereby inhibiting the self-bleaching of ECDs. In this study, phosphate glass is used as an electrolyte to construct high-energy barriers. Sodium ions in phosphate glass absorb external heat to cross energy barriers and become conductive charge carriers. In this case, the electrochromism of ECDs is allowed. On the contrary, after the absorbed heat energy is released, sodium ions are immediately trapped by oxygen ions in the PO4 unit, becoming frozen ions. At this point, the electrochromization of ECDs is prohibited. Based on the ionic conductive feature of phosphate glass, ECDs absorb heat and are colored by applying an electric field first. Then, ECDs release the thermal energy and the sodium ions transport in the electrolyte is blocked to cut off the self-bleaching pathway. The prepared inorganic all-solid-state ECDs maintained the colored state for several months using the method mentioned above, which solved the problem of the poor open-circuit memory of ECDs.

miR-125b-5p delivered by adipose-derived stem cell exosomes alleviates hypertrophic scarring by suppressing Smad2.

Background: Hypertrophic scarring is the most serious and unmet challenge following burn and trauma injury and often leads to pain, itching and even loss of function. However, the demand for ideal scar prevention and treatment is difficult to satisfy. We aimed to discover the effects and mechanisms of adipose-derived stem cell (ADSC) exosomes in hypertrophic scarring. Methods: ADSC exosomes were isolated from the culture supernatant of ADSCs and identified by nanoparticle tracking analysis, transmission electron microscopy and western blotting. The effect of ADSC exosomes on wound healing and scar formation was detected by the wound model of BALB/c mice. We isolated myofibroblasts from hypertrophic scar tissue and detected the cell viability, proliferation and migration of myofibroblasts. In addition, collagen formation and fibrosis-related molecules were also detected. To further disclose the mechanism of ADSC exosomes on fibrosis in myofibroblasts, we detected the expression of Smad2 in hypertrophic scar tissue and normal skin and the regulatory mechanism of ADSC exosomes on Smad2. Injection of bleomycin was performed in male BALB/c mice to establish an in vivo fibrosis model while ADSC exosomes were administered to observe their protective effect. The tissue injury of mice was observed via hematoxylin and eosin and Masson staining and related testing. Results: In this study, we found that ADSC exosomes could not only speed up wound healing and improve healing quality but also prevent scar formation. ADSC exosomes inhibited expression of fibrosis-related molecules such as α-smooth muscle actin, collagen I (COL1) and COL3 and inhibited the transdifferentiation of myofibroblasts. In addition, we verified that Smad2 is highly expressed in both hypertrophic scar tissue and hypertrophic fibroblasts, while ADSC exosomes downregulated the expression of Smad2 in hypertrophic fibroblasts. Further regulatory mechanism analysis revealed that microRNA-125b-5p (miR-125b-5p) is highly expressed in ADSC exosomes and binds to the 3' untranslated region of Smad2, thus inhibiting its expression. In vivo experiments also revealed that ADSC exosomes could alleviate bleomycin-induced skin fibrosis and downregulate the expression of Smad2. Conclusions: We found that ADSC exosomes could alleviate hypertrophic scars via the suppression of Smad2 by the specific delivery of miR-125b-5p.

Large scale production of highly-qualified graphene by ultrasonic exfoliation of expanded graphite under the promotion of (NH4)2CO3 decomposition.

Highly-qualified graphene was prepared by the ultrasonic exfoliation of commercial expanded graphite (EG) under the promotion of (NH4)2CO3 decomposition. The yield of graphene from the first exfoliation is 7 wt%, and it can be increased to more than 65 wt% by repeated exfoliations. Atomic force microscopy, x-ray photoelectron spectroscopy and Raman analysis show that the as-prepared graphene only has a few defects or oxides, and more than 95% of the graphene flakes have a thickness of ~1 nm. The electrochemical performance of the as-prepared graphene is comparable to reduced graphene oxide in the determination of dopamine (DA) from the mixed solution of ascorbic acid, uric acid and DA. These results show that the decomposition of (NH4)2CO3 molecules in the EG layers under ultrasonication promotes the exfoliation of graphite and provides a low-priced route for large scale production of highly-quality graphene.

Lubiprostone decreases mouse colonic inner mucus layer thickness and alters intestinal microbiota.

BACKGROUND: Lubiprostone has been used to treat constipation through its effects to stimulate Cl(-) secretion, resulting in water and electrolyte secretion. AIM: Potential associated changes in intestinal mucus and the colonizing bacteria (microbiome) have not been studied. As mucus obstructions may play a role in cystic fibrosis, the hypothesis that lubiprostone alters intestinal mucus and the microbiome was investigated. METHODS: Ion transport studies were performed ex vivo. For mucus and microbiome studies, mice were gavaged daily with lubiprostone or vehicle. Mucin from intestinal sections was analyzed in Carnoy's fixed tissues stained with Alcian blue. Microbiome composition was analyzed by 16S rRNA gene-based sequencing. RESULTS: Lubiprostone stimulated short circuit current in all mouse intestinal segments after both serosal and mucosal additions, albeit at lower concentrations in the latter. Current was Cl-dependent and blocked by mucosal diphenylcarboxylic acid, serosal bumetanide, and serosal Ba(++). The CFTR inhibitor CFTRinh172 had a marginal effect. Mucus near epithelial cells (inner layer mucus) was not present in the small intestine of any mice. Proximal colon inner mucus layer was thicker in ∆F/∆F compared with +/∆F and +/+ mice. Lubiprostone decreased inner mucus layer thickness in both proximal and distal colon of all mice. Furthermore, lubiprostone altered the intestinal microbiome by increasing abundance of Lactobacillus and Alistipes. CONCLUSIONS: Lubiprostone activates non-CFTR Cl(-) secretion and alters the colonic inner mucus layer, which is associated with changes in the composition of the enteric microbiome.

Different extraction pretreatments significantly change the flavonoid contents of Scutellaria baicalensis.

CONTEXT: Scutellaria baicalensis Georgi (Labiatae) is one of the most commonly used medicinal herbs, especially in traditional Chinese medicine. However, compared to many pharmacological studies of this botanical, much less attention has been paid to the quality control of the herb's pretreatment prior to extract preparation, an issue that may affect therapeutic outcomes. OBJECTIVE: The current study was designed to evaluate whether different pretreatment conditions change the contents of the four major flavonoids in the herb, i.e., two glycosides (baicalin and wogonoside) and two aglycones (baicalein and wogonin). MATERIALS AND METHODS: A high-performance liquid chromatography assay was used to quantify the contents of these four flavonoids. The composition changes of four flavonoids by different pretreatment conditions, including solvent, treatment time, temperature, pH value and herb/solvent ratio were evaluated. RESULTS: After selection of the first order time-curve kinetics, our data showed that at 50 °C, 1:5 herb/water (in w/v) ratio and pH 6.67 yielded an optimal conversion rate from flavonoid glycosides to their aglycones. In this optimized condition, the contents of baicalin and wogonoside were decreased to 1/70 and 1/13, while baicalein and wogonin were increased 3.5- and 3.1-fold, respectively, compared to untreated herb. DISCUSSION AND CONCLUSION: The markedly variable conversion rates by different pretreatment conditions complicated the quality control of this herb, mainly due to the high amount of endogenous enzymes of S. baicalensis. Optimal pretreatment conditions observed in this study could be used obtain the highest level of desired constituents to achieve better pharmacological effects.

[Proanthocyanidins alleviate lipopolysaccharide-induced inflammatory response by up-regulating SIRT1 expression and inhibiting NF-κB pathway in mouse RAW264.7 macrophages].

Objective To investigate the role of proanthocyanidins (PC) in lipopolysaccharide (LPS)-induced inflammatory response and its possible mechanism in RAW264.7 macrophages. Methods RAW264.7 macrophages were cultured and treated with PBS and different concentrations of PC for 24 hours, followed by 1 µg/mL LPS for 6 hours. Real-time PCR was used to detect the mRNA expression of interleukin1ß (IL-1ß), IL-6, monocyte chemoattractant protein 1 (MCP-1), tumor necrotic factor α (TNF-α), IL-4 and arginase 1 (Arg1) in RAW264.7 macrophages. Flow cytometry was used to detect the effects of PBS group, LPS group and PC combined with LPS group on M1 and M2 polarization of macrophages. The protein expressions of silenced information regulator 1 (SIRT1), nuclear factor kappa B p65(NF-κB p65) and acetylated NF-κB p65 (Ace-p65) were detected by Western blot analysis after different concentrations of PC treatment. Co-immunoprecipitation assay was used to detect the binding effect of SIRT1 to NF-κB p65 in macrophages treated with PC. Results Compared with PBS group, the mRNA expression of macrophage pro-inflammatory cytokines IL-1ß, IL-6, MCP-1 and TNF-α decreased and the mRNA expression of anti-inflammatory factors IL-4 and Arg1 increased in PC group. Compared with LPS group, PC combined with LPS group could significantly inhibit M1 polarization and promote M2 polarization of macrophages. With the increase of PC concentration, the expression of SIRT1 was up-regulated, and NF-κB p65 protein did not change significantly. The expression of Ace-p65 protein decreased significantly when treated with high concentration of PC. Conclusion PC can significantly alleviate the LPS-induced inflammatory response by up-regulating the expression of SIRT1 and inhibiting NF-κB pathway in RAW264.7 macrophages.

Extracellular vesicles from 3D cultured dermal papilla cells improve wound healing via Krüppel-like factor 4/vascular endothelial growth factor A -driven angiogenesis.

Background: Non-healing wounds are an intractable problem of major clinical relevance. Evidence has shown that dermal papilla cells (DPCs) may regulate the wound-healing process by secreting extracellular vesicles (EVs). However, low isolation efficiency and restricted cell viability hinder the applications of DPC-EVs in wound healing. In this study, we aimed to develop novel 3D-DPC spheroids (tdDPCs) based on self-feeder 3D culture and to evaluate the roles of tdDPC-EVs in stimulating angiogenesis and skin wound healing. Methods: To address the current limitations of DPC-EVs, we previously developed a self-feeder 3D culture method to construct tdDPCs. DPCs and tdDPCs were identified using immunofluorescence staining and flow cytometry. Subsequently, we extracted EVs from the cells and compared the effects of DPC-EVs and tdDPC-EVs on human umbilical vein endothelial cells (HUVECs) in vitro using immunofluorescence staining, a scratch-wound assay and a Transwell assay. We simultaneously established a murine model of full-thickness skin injury and evaluated the effects of DPC-EVs and tdDPC-EVs on wound-healing efficiency in vivo using laser Doppler, as well as hematoxylin and eosin, Masson, CD31 and α-SMA staining. To elucidate the underlying mechanism, we conducted RNA sequencing (RNA-seq) of tdDPC-EV- and phosphate-buffered saline-treated HUVECs. To validate the RNA-seq data, we constructed knockdown and overexpression vectors of Krüppel-like factor 4 (KLF4). Western blotting, a scratch-wound assay, a Transwell assay and a tubule-formation test were performed to detect the protein expression, cell migration and lumen-formation ability of KLF4 and vascular endothelial growth factor A (VEGFA) in HUVECs incubated with tdDPC-EVs after KLF4 knockdown or overexpression. Dual-luciferase reporter gene assays were conducted to verify the activation effect of KLF4 on VEGFA. Results: We successfully cultured tdDPCs and extracted EVs from DPCs and tdDPCs. The tdDPC-EVs significantly promoted the proliferation, lumen formation and migration of HUVECs. Unlike DPC-EVs, tdDPC-EVs exhibited significant advantages in terms of promoting angiogenesis, accelerating wound healing and enhancing wound-healing efficiency both in vitro and in vivo. Bioinformatics analysis and further functional experiments verified that the tdDPC-EV-regulated KLF4/VEGFA axis is pivotal in accelerating wound healing. Conclusions: 3D cultivation can be utilized as an innovative optimization strategy to effectively develop DPC-derived EVs for the treatment of skin wounds. tdDPC-EVs significantly enhance wound healing via KLF4/VEGFA-driven angiogenesis.